Immunogens against gonadotropin releasing hormone

ABSTRACT

Immunogenic compositions capable of generating an immune response in mammals against GnRH are disclosed. The immunogenic compositions are effective in methods of treating gonadotropin and gonadal steroid hormone dependent diseases and immunological contraception of mammals.

This application is a continuation of application Ser. No. 08/188,223,filed on Jan. 27, 1994 now U.S. Pat. No. 5,688,506.

BACKGROUND OF THE INVENTION

Gonadotropin Releasing Hormone ("GnRH", also known as LuteinizingHormone Releasing Hormone, or "LHRH"), is of central importance to theregulation of fertility. Johnson M., Everitt B. Essential Reproduction,3rd Edn. Blackwell Scientific Publications, 1988. In males and females,GnRH is released from the hypothalamus into the bloodstream and travelsvia the blood to the pituitary, where it induces the release of thegonadotropins, luteinizing hormone and follicle stimulating hormone, bygonadotroph cells. These two gonadotropins, in turn, act upon thegonads, inducing steroidogenesis and gametogenesis. Steroids releasedfrom the gonads into the circulation subsequently act upon varioustissues.

The gonadotropin hormonal cascade can be halted by neutralization of thebiological activity of GnRH. Fraser H. M. Physiological Effects ofAntibody to Leutenizing Hormone Releasing Hormone. In: PhysiologicalEffects of Immunity Against Reproductive Hormones, Edwards and Johnson,Eds. Cambridge University Press, 1976. As a consequence of GnRHneutralization, the gonadotropins and gonadal steroids are not releasedinto the blood and their biological activities are thereby eliminated.By eliminating the biological activity of GnRH, the hormonal regulationof fertility is interrupted and gametogenesis ceases. GnRHneutralization halts the production of gametes. GnRH neutralization isthus an effective means of contraception.

A number of important diseases are affected by gonadotropins and gonadalsteroid hormones, particularly the gonadal steroids. Such diseasesinclude breast cancer, uterine and other gynecological cancers,endometriosis, uterine fibroids, prostate cancer and benign prostatichypertrophy, among others. Removal of the gonadal steroid hormonalstimuli for these diseases constitutes an important means of therapy. Aneffective method of accomplishing this is by neutralizing GnRH, theconsequence of which is the elimination of gonadal steroids that induceand stimulate these diseases. McLachlan R. I., Healy D. L., Burger G. B.1986. Clinical Aspects of LHRH Analogues in Gynecology: a Review,British Journal of Obstetrics and Gynecology, 93:431-454. Conn P. M.,Crowley W. F. 1991. Gonadotropin-Releasing Hormone and Its Analogs, NewEngland Journal of Medicine. 324:93-103. Filicori M., Flamigni C. 1988.GnRH Agonists and Antagonists, Current Clinical Status. Drugs. 35:63-82.

One effective means of neutralizing GnRH is the induction orintroduction of anti-GnRH antibodies in the host or patient. Suchantibodies can be induced by active immunization with GnRH immunogens orby passive immunization by administering anti-GnRH antibodies. Fraser H.M. Physiological Effects of Antibody to Leutenizing Hormone ReleasingHormone. In: Physiological Effects of Immunity Against ReproductiveHormones, Edwards and Johnson, Eds. Cambridge University Press, 1976.Since anti-GnRH antibodies can neutralize the biological activity ofGnRH, immunization constitutes an important approach towards treatingdiseases dependent upon gonadal steroids and other reproductive hormonesas well as a means to regulate mammalian fertility.

GnRH has the same amino acid sequence in all mammals(pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-GlyNH₂) (SEQ ID NO.: 1 in theSequence Listing), thus a single immunogen would be effective in allmammalian species, including humans. Active immunization against GnRH,however, has not been practicable due to deficiencies associated withthe GnRH immunogens. The prior art anti-GnRH immunogens are not ofsufficient potency, and therefore must be administered repeatedly toinduce effective levels of anti-GnRH antibodies. In addition, they havenot proven to be reliable, in terms of inducing anti-GnRH antibodies inan acceptable portion of the immunized population.

SUMMARY OF THE INVENTION

The present invention, concerns improved immunogens against GnRH thatinduce neutralizing titers of anti-GnRH antibodies in response to asingle administration of immunogen in all of the immunized populationsthat we have studied. The immunogens of the invention may thus be usedto treat steroid dependent diseases and may also be used asimmunocontraceptives to regulate fertility.

The immunogens of the present invention are peptides composed of twofunctional regions: the immunomimic region and a spacer region. Thefunction of the immunomimic which immunologically crossreacts with GnRHis to induce antibodies that bind to the targeted hormone. The spacerelement of the peptide serves as a link through which the immunomimic isattached to an immunological carrier, such as diphtheria toxoid ("DT")and also affects the immune response generated by the vaccinated mammalagainst the immunomimic. For example, in a specific embodiment of theinvention, the immunogen peptide has the sequence:pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-Arg-Pro-Pro-Pro-Pro-Cys (SEQ IDNO: 2 in the Sequence Listing). In this ("GnRH(1-10)-Arg10") peptide,the sequence pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-- (SEQ ID NO: 3 inthe Sequence Listing), comprises the immunomimic of GnRH. The remainderof the peptide's sequence, --Arg-Pro-Pro-Pro-Pro-Cys (SEQ ID NO: 4 inthe Sequence Listing), constitutes the spacer, which is attached toamino acid number 10 of the GnRH immunomimic.

A preferred embodiment of the invention concerns two peptideimmunomimics of GnRH that are associated with four spacer sequences.Methods of coupling these peptides to immunological carriers, such asDT, to yield anti-GnRH immunogens are provided. The immunogens may beused singly or in combination to induce anti-GnRH antibody responses inthe vaccinated mammal. As compared to the prior art anti-GnRHimmunogens, the immunogens of the present invention induce abiologically effective immune response following a single administrationof immunogen in all of the immunized animals tested. The immunogens canbe administered in different physical forms, including soluble andprecipitate. The immunomimic spacer peptides of this invention can becoupled to immunological carriers over a wide range of peptide tocarrier substitution ratios and yield effective immunogens.

The invention also concerns methods of treating gonadotropin and gonadalsteroid hormone dependent diseases and cancers by immunization with theimmunogens of the invention. A specific embodiment of the inventionconcerns a method of immunological contraception in mammals comprisingthe administration of the inventive immunogens.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Depicts anti-GnRH antibody responses to the administration ofthe inventive immunogens comprising peptides 1-4 and the comparativeprior art anti-GnRH immunogen, peptide 5 as measured by mean antigenbinding capacities ("ABC") in pico moles per milliliter with respect todays after immunization in immunized rabbits.

FIG. 2: Depicts the antibody response to immunization with an immunogencomprising a mixture of peptides 3 and 4 as measured by mean ABC withrespect to days after immunization.

FIG. 3: Depicts the results of immunizations in mice as measured by amean ABC with respect to days after immunization after immunization withfractions of a preparation of peptide 2 immunogens fractionated on thebasis of solubility.

FIG. 4: Depicts antibody responses in mice as measured by mean ABC withrespect to days after immunization when immunized with variousconjugates of peptides 1 and 2 at different peptide: DT substitutionratios.

FIG. 5: Depicts antibody responses of male rabbits as measured by meanABC with respect to days after immunization when immunized with amixture of conjugates of peptides 1 and 2. Serum testosterone levels inthese male rabbits over the course of the immunization test period areshown.

DETAILED DESCRIPTION OF THE INVENTION EXAMPLE 1

Peptides with the amino acid sequences listed in Table 1 weresynthesized and prepared by standard solid phase synthesis methods. Eachpeptide was characterized as to amino acid content and purity.

                                      TABLE 1                                     __________________________________________________________________________    Peptide                                                                            Designation                                                                             Amino Acid Sequence                                            __________________________________________________________________________    1    GnRH(1-10)-Ser1                                                                         Cys--Pro--Pro--Pro--Pro--Ser--Ser--Glu--His--                      Trp--Ser--Tyr--Gly--Leu--Arg--Pro--                                           Gly(NH.sub.2)(SEQ ID NO: 5 in the                                             Sequence Listing)                                                           2 GnRH(1-10)-Ser10 pGlu--His--Trp--Ser--Tyr--Gly--Leu--Arg--                    Pro--Gly--Ser--Ser--Pro--Pro--Pro--Pro--Cys                                   (SEQ ID NO: 6 in the Sequence                                                 Listing)                                                                    3 GnRH(1-10)-Arg1 Cys--Pro--Pro--Pro--Pro--Arg--Glu--His--Trp--                               Ser--Tyr--Gly--Leu--Arg--Pro--Gly(NH.sub.2)                     (SEQ ID NO: 7 in the Sequence                                                 Listing)                                                                    4 GnRH(1-10)-Arg10 pGlu--His--Trp--Ser--Tyr--Gly--Leu--Arg--                    Pro--Gly--Arg--Pro--Pro--Pro--Pro--Cys                                        (SEQ ID NO: 2 in the Sequence                                                 Listing)                                                                  __________________________________________________________________________

Each of peptides 1-4 contains an immunomimic of GnRH that is eitherpreceded by or followed by a spacer. Two immunomimics of GnRH were used:pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-- (SEQ ID NO: 3 in the SequenceListing), (peptides 2 and 4 Table 1) wherein the spacer was attachedthrough the carboxy terminal end of GnRH (amino acid #10); and,--Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly(NH₂) (SEQ ID NO: 8 in theSequence Listing), (peptides 1 and 3 Table 1) wherein the spacer wasattached at the amino terminal end of GnRH (amino acid #1).

The four spacers set forth in Table 2 were used.

                  TABLE 2                                                         ______________________________________                                        Spacer Designation                                                                          Amino Acid Sequence                                             ______________________________________                                        Ser 1         Cys--Pro--Pro--Pro--Pro--Ser--Ser--                                (SEQ ID NO: 9 in the Sequence Listing)                                       Ser 10 --Ser--Ser--Pro--Pro--Pro--Pro--Cys                                     (SEQ ID NO: 10 in the Sequence Listing)                                      Arg 1 Cys--Pro--Pro--Pro--Pro--Arg--                                           (SEQ ID NO: 11 in the Sequence Listing)                                      Arg 10 --Arg--Pro--Pro--Pro--Pro--Cys                                          (SEQ ID NO: 4 in the Sequence Listing)                                     ______________________________________                                    

The numerals 1 and 10 in the spacer designation refer to the GnRH aminoacid number to which the spacer is attached. While these spacer regionsof the molecules have been set forth separately in Table 2, in thepreferred embodiment of the invention the peptide is synthesized as onecontinuous peptide sequence molecule.

Each of these peptides 1-4 of Table 1 was conjugated to amino groupspresent on a carrier such as Diphtheria Toxoid ("DT") via the terminalpeptide cysteine residue utilizing heterobifunctional linking agentscontaining a succinimidyl ester at one end and maleimide at the otherend of the linking agent.

To accomplish the linkage between any of the Peptides 1-4 above and thecarrier, the cysteine of the peptide was first reduced. The dry peptidewas dissolved in 0.1 M sodium phosphate buffer (degassed), pH 8.0, witha thirty molar excess of dithiothreitol ("DTT"). The solution wasstirred under a water saturated nitrogen gas atmosphere for three hoursat room temperature. An additional 15 molar excess DTT was added and themixture was stirred an additional hour at room temperature under watersaturated nitrogen gas. The peptide containing reduced cysteine wasseparated from the other components by chromatography at 4° C. over aG10 Sephadex column equilibrated with 0.2 M acetic acid. The peptide waslyophilized and stored under vacuum until used.

The DT was activated for coupling to the peptide by treatment with theheterobifunctional linking agent epsilon-maleimidocaproic acidN-hydroxysuccinimide ester ("EMCS"), in proportions sufficient toachieve activation of approximately 25 free amino groups per 10⁵molecular weight of DT. In the specific instance of DT, this amounted tothe addition of 6.18 mg of EMCS (purity 98%) to each 20 mg of DT.

Activation of DT was accomplished by dissolving each 20 mg aliquot of DTin 1 ml of 0.5 M sodium phosphate buffer, pH 6.6. Aliquots of 6.18 mgEMCS were dissolved into 0.2 ml of dimethylformamide. Under darkenedconditions, the EMCS was added dropwise in 50 microliter ("μl") amountsto the DT with stirring. After 90 minutes incubation at room temperaturein darkness, the mixture was chromatographed at 4° C. on a G50 Sephadexcolumn equilibrated with 0.1 M sodium citrate buffer, pH 6.0, containing0.1 mM ethylenediaminetetraacetic acid disodium salt ("EDTA").(Column=1.5×120 cm; flow rate=8 ml/hr; fraction size=2 ml). Thefractions' A₂₆₀ were determined using a spectrophotometer, enabling thefractions containing DT to be identified.

Fractions containing the EMCS activated DT were pressure concentratedover a PM 10 ultrafiltration membrane under nitrogen gas in conditionsof darkness. The protein content of the concentrate was determined bythe BCA method (PIERCE, Ill., USA). The EMCS content of the carrier wasdetermined by incubation of the activated DT with cysteine-HCl followedby reaction with 100 μl of 10 mM Elman's Reagent (5, 5, dithio-bis(2-nitrobenzoic acid)). The optical density difference between a blanktube containing cysteine-HCl and the sample tube containing cysteine-HCland carrier was translated into EMCS group content by using themolecular extinction coefficient of 13.6×10³ for 5-thio-2-nitro-benzoicacid at 412 nm.

The reduced cysteine content ("-SH") of the peptide was also determinedutilizing Elman's Reagent. Approximately 1 mg of peptide was dissolvedin 1 ml of nitrogen gas saturated water and a 0.1 ml aliquot of thissolution was reacted with Elman's Reagent. Utilizing the molarextinction coefficient of 5-thio-2-nitro-benzoic acid (13.6×10³), thefree cysteine -SH was calculated.

The reduced peptide was then coupled to the activated DT. An amount ofpeptide containing sufficient free -SH to react with a selectedproportion of the EMCS activated amino groups on the DT was dissolved in0.1 M sodium citrate buffer, pH 6.0, containing 0.1 mM EDTA, and addeddropwise to the EMCS activated DT under darkened conditions. After allthe peptide solution had been added to the activated DT, the mixture wasincubated overnight in the dark under a water saturated nitrogen gasatmosphere at room temperature.

The conjugate of the peptide linked to DT via EMCS was separated fromother components of the mixture by low pressure chromatography at 4° C.over a G50 Sephadex column equilibrated with 0.2 M ammonium bicarbonate(column=1.5×120 cm, flow rate=1.8 ml/15 min., fraction size=1.8 ml). Theconjugate eluted in the column void volume (detected by A₂₈₀measurements) and was lyophilized and stored desiccated at -20° C. untilused.

The conjugate may be characterized as to peptide content by a number ofmethods known to those skilled in the art including weight gain, aminoacid analysis, etc. Various substitution ratios of peptide to DT wereaccurately and reproducibly obtained by (1) varying the quantity of EMCSadded to activate the DT, and/or, (2) varying the quantity of reducedpeptide added to the EMCS activated DT. For example, the activation ofDT with a ratio of 31 moles EMCS to 1 mole of 100,000 molecular weightDT adds 12±2 EMCS groups per 100,000 molecular weight of DT. Theaddition of 14 peptide groups per 100,000 molecular weight of thisactivated DT resulted in a substitution ratio of 12±2 peptides per100,000 molecular weight of DT. Conjugates of Peptides 1-4 to DTproduced by these methods were determined by amino acid analysis to have4-30 moles of peptide per 10⁵ MW of DT. All of the conjugates wereconsidered suitable as immunogens for immunization of test animals.

EXAMPLE 2

For comparative purposes a prior art GnRH immunogen ("peptide 5") wasconstructed wherein the peptide immunomimic of GnRH did not contain aspacer element. Peptide 5 had the sequence:Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-GlyNH₂ (SEQ ID NO: 8 in the SequenceListing).

The peptide was activated with m-Maleimidobenzoyl N-HydroxysuccinimideEster ("MBS"). 20.0 mg of [glu 1]-GnRH were dissolved in 1.0 ml ofN,N-Dimethylformamide ("DMF"). To this solution was added 5.31 mg MBSdissolved in 1.0 ml DMF. The combined solution was stirred overnight atroom temperature in the dark.

40.0 mg of DT was dissolved in 10.0 ml of Sodium Carbonate Buffer (0.2M, pH=9.0), containing 2.2 mg of 2-Iminothiolane HCl ("2-IT"). Thesolution containing the MBS-activated GnRH was then slowly added to theDT/2-IT solution, and the mixture was stirred slowly for 8 hours at roomtemperature in the dark.

The conjugate was purified by column chromatography over Sephadex G50(column: 1.5×100 cm; buffer: Ammonium Bicarbonate, 0.2 M; fractions: 2.6ml, every 15 minutes) with identification of the fractions containingconjugate by spectrophotometry (A₂₅₄). G50 purified conjugate waslyophilized and stored desiccated at -20° C. until used. The peptide DTsubstitution ratio of the Immunogen 5 conjugate was determined by aminoacid analysis to be 13 peptides per 10⁵ molecular weight of DT.

EXAMPLE 3

Different groups of female rabbits were each immunized with one of theconjugates, peptides 1-5 of Examples 1 and 2. Each conjugate wasdissolved to a concentration of 2.0 mg/ml in phosphate buffered saline(0.2 M, pH=7.2) containing 200 μg/ml of norMDP adjuvant. The conjugatescomprising peptides 1,2,3 and 4 of Example 1 did not completely dissolvein the buffer; the conjugate of peptide 5 of Example 2 did completelydissolve in the buffer. Each mixture was emulsified with an equal volumeof Squalene-Arlacel (4:1 ratio, volume of Squalene: volume of Arlacel)to prepare an immunogen formulation which contained 1.0 mg/ml conjugateand 100 μg/ml norMDP. 1.0 ml of immunogen was injected into each rabbit,administered into the rear leg muscles (2 sites, 0.5 ml/site), on day 0of the test. Blood was collected from each rabbit prior to immunizationon day 0, and on selected days thereafter. Serum was prepared from eachblood sample and stored frozen at -20° C. until utilized in assays todetermine the presence of anti-GnRH antibodies.

A liquid phase Radioimmunoassay (RIA) was used to detect and quantifyanti-GnRH antibodies. In the RIA, 0.04, 0.2, 1.0 or 5.0 μl aliquots ofantiserum were incubated with approximately 150 fmole of 3H labeled GnRH(specific activity=53.2 Ci/mmole) in a total volume of 400 μl. Dilutionswere made in FTA Hemagglutination Buffer (BBL, Becton DickinsonMicrobiology Systems, MD, USA) containing 1% bovine serum albumin. Theantisera were incubated with labeled hormone for 2 hours at roomtemperature. A 0.1 ml aliquot of heat inactivated (56° C., 30 min) fetalcalf serum (cooled to 2-8° C.) was then added to each tube, followingwhich the antibody-hormone complexes were precipitated by the additionof 0.5 ml of 25% polyethylene glycol (MW=8,000 gm/mole) (cooled to 2-8°C.). The precipitates were pelleted by centrifugation (30 minutes at1500×g), the supernatants were discarded, and the pellets were countedby liquid scintillation counting to measure the quantity ofradioactivity contained therein. Antigen binding capacities (ABC) foreach antiserum were then determined from the amount of radioactivehormone precipitate after subtraction of nonspecific background binding(determined by preincubation of the antisera dilution with excessamounts (˜10⁵ fold) of the hormone). Inhibition of the antisera with theexcess quantity of unlabeled hormone also established the specificity ofthe antisera for GnRH. Serum taken from the rabbits prior toimmunization served as nonimmunized (normal) controls.

The mean ABCs measured in the sera from rabbits immunized with theconjugated peptides of Examples 1 and 2 are shown in Table 3 and inFIG. 1. As the results show, a single administration of the immunogenscomprising peptides 1,2,3 and 4 of Example 1 induced rapid and potentantibody responses against GnRH.

                                      TABLE 3                                     __________________________________________________________________________    RABBIT ANTI-GNRH ANTIBODY RESPONSES INDUCED                                     BY ONE ADMINISTRATION OF PEPTIDE CONJUGATE                                      Peptide:DT                                                                  Substitution Rabbit Sera ABC (mean) [pmoles/ml]                             Peptide                                                                           Ratio Day 0                                                                             Day 14                                                                            Day 21                                                                            Day 28                                                                            Day 36                                                                            Day 44                                                                            Day 56                                                                            Day 73                                                                            Day 85                                                                            Day 105                         __________________________________________________________________________      1 13 0 0.30 10.83 22.63 57.23 68.93 72.13 61.23 58.73 54.03                   2 13 0 0.27 7.52 19.83 57.63 77.83 78.73 60.83 47.90 24.93                    5 13 0 0  0 1.78  1.60 1.51 2.00 2.10                                           Day 0 Day 15 Day 24 Day 31  Day 44 Day 59  Day 79 Day 101                   3 11 0 1.53 24.59 58.31  102.71 118.16  120.99 61.00                          4 13 0 1.77 8.90 26.03  42.88 38.25  38.30 24.35                            __________________________________________________________________________     n = 5 rabbits for Peptides 1, 2, 3 and 4. n = 6 rabbits for peptide 5.   

By comparison, the anti-GnRH response induced by a single administrationof the peptide 5 immunogen of Example 2 induced a minimal response. Thisis not because the conjugate constructed with peptide 5 is a poorimmunogen; when administered in additional booster immunizations severalweeks after the first immunization, the peptide 5 conjugate induceseffective levels of anti-GnRH antibodies (of approximately 12-18pmole/ml ABC). In this regard, the peptide 5 conjugate behaves similarlyto standard GnRH immunogens. However, the conjugate constructed withpeptide 5 requires more than one administration, induces lower levels ofanti-GnRH antibodies, and takes a longer time to elicit effectiveantibody levels than do the conjugates of peptides 1-4 of Example 1.

These results also demonstrate the critical contribution of the spacerto the immunogenicity of peptides 1,2,3 and 4 of Example 1. Peptide 5bears the same immunomimic of GnRH as peptides 1 and 3, yet peptide 5 isinferior as an immunogen. This is because peptide 5 does not contain aspacer sequence, which is present in peptides 1 and 3. Thus, thepresence of the spacers in peptides 1,2,3 and 4 of Example 1 makes acritical contribution to their enhanced immunogenicity.

EXAMPLE 4

Conjugates comprising peptides 3 and 4 of Example 1 were mixed 1:1 togive a protein concentration of 2.0 mg/ml in PBS. The mix was thenprepared as immunogen and injected into rabbits, as in Example 3. Thesera were tested for anti-GnRH antibody by the RIA of Example 3. Theresults are shown in Table 4 and FIG. 2.

                                      TABLE 4                                     __________________________________________________________________________    RABBIT ANTI-GNRH RESPONSES INDUCED BY                                           ONE ADMINISTRATION OF A MIXTURE OF PEPTIDE CONJUGATES                         ABC (mean ± s.e.) [moles/ml]                                             Day of Test                                                                         0 15   24    31    44    59    79    101                                __________________________________________________________________________      ABC 0 4.6 ± 0.7 21.6 ± 3.3 49.0 ± 9.9 77.8 ± 13.0 86.0 ±                                                21.0 74.3 ± 22.0 43.0 ±                                                 12.0                               __________________________________________________________________________

As can be seen from Table 4, effective levels of antibody were inducedby the combined administration of the peptide 3 and 4 conjugates. Bothpeptide components contributed almost equally to the induction of theanti-GnRH antibodies, as shown by antibody specificity testing. The GnRH(1-10)-Arg1 peptide induced antibodies directed predominantly againstthe carboxy terminal end of GnRH, while the GnRH(1-10)-Arg10 peptideinduced antibodies directed against the amino terminal end of GnRH.Thus, conjugates comprising these peptides can be mixed to yieldimmunogens that induce antibodies against both ends of the targetmolecule.

EXAMPLE 5

When the peptides of Example 1 are conjugated to DT and prepared asdescribed in Example 1, a proportion of the product is present as aprecipitate. The formation of the precipitate is dependent upon variousphysical parameters, including concentration of conjugate, pH and saltconcentration. We prepared a conjugate of peptide 2 of Example 1 to DTas described in Example 1. From this we prepared three fractions ofconjugate, based upon solubility. The conjugate was stirred in 0.01 Mphosphate buffer pH=7.2 and the insoluble material was collected bycentrifugation as Fraction #1. To the soluble material we added NaCl (to0.5 M) and adjusted the pH to 6.0 with 0.1 M HCl, which yieldedadditional precipitate that we collected as Fraction #2. The remainingsoluble material served as Fraction #3. Each fraction was lyophilized.The percent recoveries (from the 15 mg of starting material) were:Fraction-1, 36%; Fraction-2, 15%; Fraction-3, 27%; lost, 22%.

Each of the fractions 1-3 were injected into a group of mice, at 6mice/group. (100 μg conjugate/mouse, with 25 μg nMDP, in 0.1 ml of a 1:1mixture of FTA buffer (containing conjugate+adjuvant) tosqualene-arlacel, i.p.). The mice received a single injection ofimmunogen, after which sera samples were obtained at intervals andtested for anti-GnRH antibody by the RIA of Example 3. The results ofthis test are shown in Table 5 and in FIG. 3.

                                      TABLE 5                                     __________________________________________________________________________    ANTI-GnRH RESPONSES OF MICE TO                                                  SOLUBILITY FRACTIONS OF CONJUGATE                                           Conjugate                                                                          ABC (mean ± s.e.) [moles/ml]                                          Fraction                                                                           Day 0                                                                             Day 14                                                                             Day 21                                                                             Day 28                                                                             Day 36                                                                             Day 45                                                                             Day 56                                      __________________________________________________________________________    1    0   1.7 ± 0.3                                                                       4.5 ± 0.4                                                                       4.6 ± 0.4                                                                       5.6 ± 0.4                                                                       5.9 ± 0.5                                                                       5.6 ± 0.4                                  2 0 1.7 ± 0.4 4.2 ± 0.3 4.6 ± 0.2 5.7 ± 0.2 5.8 ± 0.2                                          5.8 ± 0.2                                  3 0 1.7 ± 0.3 4.0 ± 0.3 4.5 ± 0.3 5.3 ± 0.3 5.3 ± 0.3                                          5.0 ± 0.3                                __________________________________________________________________________

As the results show, each mouse group produced equally potent anti-GnRHantibody responses. This demonstrates that despite variances in thesolubility of conjugates produced from the peptide of Example 1, thesoluble and insoluble forms can be administered as immunogens and are ofequivalent immunogenicity.

EXAMPLE 6

We constructed conjugates of peptides 1 and 2 of Example 1 to DT asdescribed in Example 1. By varying the quantities of reduced peptideadded to DT, we constructed conjugates with different peptide:DTsubstitution ratios. The substitution ratios, determined by amino acidanalysis of the conjugates are shown in Table 6:

                  TABLE 6                                                         ______________________________________                                        Conjugate   Peptide Used                                                                              Peptide:DT                                              Number (from Example 1) Substitution Ratio                                  ______________________________________                                        6.1         1           4.7                                                     6.2 1 13.1                                                                    6.3 1 25.9                                                                    6.4 2 5.1                                                                     6.5 2 12.8                                                                    6.6 2 30.1                                                                  ______________________________________                                    

Mice were immunized with each conjugate preparation. The immunizationand subsequent assay procedures were identical to those described inExample 5 (6 mice/group). The results of this test are shown in Table 7and in FIG. 4.

                                      TABLE 7                                     __________________________________________________________________________    ANTI-GnRH RESPONSES OF MICE TO PEPTIDE-CARRIER                                  CONJUGATES WITH A DIFFERENT SUBSTITUTION RATIOS                                  Peptide:DT                                                                 Conjugate Substitution ABC (mean ± s.e.) [pmoles/ml]                     number                                                                             Ratio Day 0                                                                             Day 14                                                                             Day 28                                                                             Day 45                                                                             Day 56                                                                              Day 70                                                                              Day 85                                                                              Day 105                       __________________________________________________________________________    6.1  4.7   0   0.7 ± 0.1                                                                       5.1 ± 0.4                                                                       9.8 ± 0.5                                                                       9.4 ± 0.4                                                                        10.5 ± 0.6                                                                       11.0 ± 0.8                                                                       10.0 ± 1.0                   6.1 13.1 0 1.8 ± 0.3 7.4 ± 0.6 9.7 ± 0.4 10.1 ± 0.2  12.2                                                       ± 0.2  11.9 ± 0.2                                                       11.0 ± 0.2                   6.3 25.9 0 0.4 ± 0   2.1 ± 0.5 4.9 ± 1.0 5.1 ± 1.1 4.7 ±                                                     1.3 5.7 ± 1.7 4.7 ±                                                     1.6                             6.4 5.1 0 1.7 ± 0.6 4.1 ± 0.6 4.6 ± 0.7 5.0 ± 0.6 6.8 ±                                                      0.9 7.3 ± 1.2 6.7 ±                                                     1.1                             6.5 12.8 0 1.4 ± 0.1 4.5 ± 0.2 5.4 ± 0.3 6.1 ± 0.4 7.2 ±                                                     0.2 8.4 ± 0.3 7.9 ±                                                     0.3                             6.6 30.1 0 1.1 ± 0.4 3.9 ± 0.4 4.6 ± 0.4 5.4 ± 0.4 6.6 ±                                                     0.5 7.4 ± 0.5 7.0 ±                                                     0.5                           __________________________________________________________________________

As the results show, significant anti-GnRH responses were induced byeach of the conjugate preparations. This demonstrates that the peptidesof Example 1 can be conjugated to carriers over a broad range ofpeptide:carrier substitution ratios and yield effective immunogens.

EXAMPLE 7

We constructed conjugates of peptides 1 and 2 of Example 1 to DT asdescribed in Example 1. The peptide:DT substitution ratio for peptide 1(GnRH(1-10)-Ser1) was 13.1:1 and the ratio for peptide 2(GnRH(1-10)-Ser10) was 12.8:1.

We prepared immunogen by emulsifying aqueous phase (containing a mixtureof the two conjugates plus norMDP in PBS) with oily vehicle as describedin Example 3. The oily vehicle used was Montanide ISA 703 containing1.8% aluminum monostearate. "Montanide ISA 703 AMS" is manufactured andsold by SEPPIC, Inc. (Paris, France). The final concentrations of theactive components in the immunogen were: GnRH (1-10)-Ser1-DT=0.5 mg/ml;GnRH (1-10)-Ser10-DT=0.5 mg/ml; norMDP=0.1 mg/ml. 1.0 ml of immunogenwas injected into each of 3 male rabbits, administered to the rear legmuscles (2 sites/rabbit, 0.5 ml/site), on day 0 of the test. Blood wascollected from each rabbit prior to immunization and on selected daysthereafter. Serum was prepared from each blood sample and stored frozenat -20° C. until utilized in assays to determine the presence ofanti-GnRH antibodies (as described in Example 3).

The mean ABC's measured in the sera from these three male rabbits areshown in Table 8 and in FIG. 5. As the results show, a singleimmunization with the DT conjugates of peptides 1 and 2 of Example 1 inthe Montanide ISA 703 containing 1.8% AMS rapidly induced potentantibody responses against GnRH. These anti-GnRH responses arerepresentative of responses induced by the peptide conjugates(individually or mixtures thereof) of this invention when administeredwith norMDP in an emulsion comprising equal parts aqueous phase andMontanide ISA 703 containing 1.8% AMS.

                  TABLE 8                                                         ______________________________________                                              Mean ABC                                                                  Day (pmol/ml) (± s.e.) Day Mean ABC (pmol/ml) (± s.e.)                ______________________________________                                        0     0.02 (± 0.1)                                                                            46        543 (± 85.0)                                    7 0.18 (± 0)   60   1061 (± 368.2)                                      14 3.71 (± 0.8) 74 1303.3 (± 527.6)                                     24 40.3 (± 7.7) 88 1320.7 (± 602.9)                                     32 131.5 (± 29.1) 102   1272 (± 558.1)                                  40 374.7 (± 13.1) -- --                                                  ______________________________________                                    

EXAMPLE 8

The production of gonadal steroids can be assessed as a measure ofGnRH-immunogen efficacy in immunized animals. We measured testosteronelevels in the serum samples obtained from the three male rabbits ofExample 7. The testosterone levels were determined using aradioimmunoassay kit for testosterone determination ("Coat-a-Count",purchased from Diagnostic Products Corp., Los Angeles, Calif., USA). Theresults presented in Table 9 and in FIG. 5 show the immunogen inducedlevels of anti-GnRH antibodies that totally inhibited the production oftestosterone in the male rabbits.

Testosterone was undetectable in the sera of 2 animals by day 24 of thetest, and in all 3 rabbits by day 32. The drop in testosterone serumcoincides with the rise in anti-GnRH Ab titer, as can be seen in FIG. 5.

                  TABLE 9                                                         ______________________________________                                        Testosterone Levels In Immunized Rabbits                                        Day     Mean T (ng/ml) (± s.e.)                                                                    Day   Mean T (ng/ml) (± s.e.)                    ______________________________________                                        0     0.32 (± 0.2) 46      0                                                 7 1.37 (± 0.1) 60 0                                                        14 1.21 (± 0.5) 74 0                                                       24 0.1 (± 0)  88 0                                                         32 0 102 0                                                                    40 0 -- --                                                                  ______________________________________                                    

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#/note= "Gonadotropin releasing                          hormone ( - #GnRH)"                                             - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 1                                                               (D) OTHER INFORMATION: - #/label= pGlu                                             /note= - #"pyroglutamic acid (5-oxoproline)"                    - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 10                                                              (D) OTHER INFORMATION: - #/label= GlyNH2                                           /note= - #"glycinamide"                                         - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                               - - Xaa His Trp Ser Tyr Gly Leu Arg Pro Xaa                                  1               5   - #                10                                      - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 16 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -    (iii) HYPOTHETICAL: YES                                                - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Region                                                          (B) LOCATION: 1..10                                                           (D) OTHER INFORMATION: - #/note= "immunomimic"                       - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Region                                                          (B) LOCATION: 11..16                                                          (D) OTHER INFORMATION: - #/note= "spacer"                            - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 1                                                               (D) OTHER INFORMATION: - #/label= pGlu                                             /note= - #"pyroglutamic acid (5-oxoproline)"                    - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                               - - Xaa His Trp Ser Tyr Gly Leu Arg Pro Gly Ar - #g Pro Pro Pro Pro Cys      1               5   - #                10  - #                15               - -  - - (2) INFORMATION FOR SEQ ID NO:3:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -    (iii) HYPOTHETICAL: YES                                                - -      (v) FRAGMENT TYPE: N-terminal                                        - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 1                                                               (D) OTHER INFORMATION: - #/label= pGlu                                             /note= - #"pyroglutamic acid (5-oxoproline)"                    - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..10                                                           (D) OTHER INFORMATION: - #/note= "immunomimic"                       - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                               - - Xaa His Trp Ser Tyr Gly Leu Arg Pro Gly                                  1               5   - #                10                                      - -  - - (2) INFORMATION FOR SEQ ID NO:4:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino - #acids                                                  (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -    (iii) HYPOTHETICAL: YES                                                - -      (v) FRAGMENT TYPE: C-terminal                                        - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..6                                                            (D) OTHER INFORMATION: - #/note= "spacer"                            - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                               - - Arg Pro Pro Pro Pro Cys                                                  1               5                                                              - -  - - (2) INFORMATION FOR SEQ ID NO:5:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -    (iii) HYPOTHETICAL: YES                                                - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Region                                                          (B) LOCATION: 1..7                                                            (D) OTHER INFORMATION: - #/note= "spacer"                            - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Region                                                          (B) LOCATION: 8..17                                                           (D) OTHER INFORMATION: - #/note= "immunomimic"                       - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 17                                                              (D) OTHER INFORMATION: - #/label= GlyNH2                                           /note= - #"glycinamide"                                         - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                               - - Cys Pro Pro Pro Pro Ser Ser Glu His Trp Se - #r Tyr Gly Leu Arg Pro      1               5   - #                10  - #                15               - - Xaa                                                                       - -  - - (2) INFORMATION FOR SEQ ID NO:6:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 17 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -    (iii) HYPOTHETICAL: YES                                                - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 1                                                               (D) OTHER INFORMATION: - #/label= pGlu                                             /note= - #"pyroglutamic acid (5-oxoproline)"                    - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Region                                                          (B) LOCATION: 1..10                                                           (D) OTHER INFORMATION: - #/note= "immunomimic"                       - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Region                                                          (B) LOCATION: 11..17                                                          (D) OTHER INFORMATION: - #/note= "spacer"                            - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                               - - Xaa His Trp Ser Tyr Gly Leu Arg Pro Gly Se - #r Ser Pro Pro Pro Pro      1               5   - #                10  - #                15               - - Cys                                                                       - -  - - (2) INFORMATION FOR SEQ ID NO:7:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 16 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -    (iii) HYPOTHETICAL: YES                                                - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Region                                                          (B) LOCATION: 1..6                                                            (D) OTHER INFORMATION: - #/note= "spacer"                            - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Region                                                          (B) LOCATION: 7..16                                                           (D) OTHER INFORMATION: - #/note= "immunomimic"                       - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 16                                                              (D) OTHER INFORMATION: - #/label= GlyNH2                                           /note= - #"glycinamide"                                         - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                               - - Cys Pro Pro Pro Pro Arg Glu His Trp Ser Ty - #r Gly Leu Arg Pro Xaa      1               5   - #                10  - #                15               - -  - - (2) INFORMATION FOR SEQ ID NO:8:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino - #acids                                                 (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -    (iii) HYPOTHETICAL: YES                                                - -      (v) FRAGMENT TYPE: C-terminal                                        - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..10                                                           (D) OTHER INFORMATION: - #/note= "immunomimic"                       - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 10                                                              (D) OTHER INFORMATION: - #/label= GlyNH2                                           /note= - #"glycinamide"                                         - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                               - - Gly His Trp Ser Tyr Gly Leu Arg Pro Xaa                                  1               5   - #                10                                      - -  - - (2) INFORMATION FOR SEQ ID NO:9:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino - #acids                                                  (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -    (iii) HYPOTHETICAL: YES                                                - -      (v) FRAGMENT TYPE: N-terminal                                        - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..7                                                            (D) OTHER INFORMATION: - #/note= "spacer"                            - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                               - - Cys Pro Pro Pro Pro Ser Ser                                              1               5                                                              - -  - - (2) INFORMATION FOR SEQ ID NO:10:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino - #acids                                                  (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -    (iii) HYPOTHETICAL: YES                                                - -      (v) FRAGMENT TYPE: C-terminal                                        - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..7                                                            (D) OTHER INFORMATION: - #/note= "spacer"                            - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                              - - Ser Ser Pro Pro Pro Pro Cys                                              1               5                                                              - -  - - (2) INFORMATION FOR SEQ ID NO:11:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino - #acids                                                  (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -    (iii) HYPOTHETICAL: YES                                                - -      (v) FRAGMENT TYPE: N-terminal                                        - -     (ix) FEATURE:                                                                  (A) NAME/KEY: Peptide                                                         (B) LOCATION: 1..6                                                            (D) OTHER INFORMATION: - #/note= "spacer"                            - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                              - - Cys Pro Pro Pro Pro Arg                                                  1               5                                                            __________________________________________________________________________

We claim:
 1. An immunogenic composition for regulating gonadal steroidhormone in a mammal comprising an anti-GnRH immunogenic conjugateconsisting of an immunogenic carrier and a GnRH an immunomimic peptideextended by a spacer peptide sequence effective for immune response. 2.A method of regulating mammalian gonadal steroid hormone associateddisorders or diseases comprising administering the immunogeniccomposition according to claim 1 to a patient in need thereof.
 3. Themethod according to claim 2, wherein the immunogenic composition isadministered as a single dosage.
 4. A mammalian anti-Gn RH immunogenicconjugate comprising an immunomimic peptide attached to an immunogeniccarrier by a spacer which is seven amino acids long.